Polymorphonuclear Leucocytes Defective in Oxidative Metabolism Inhibit in Vitro Growth of Plasmodium falciparum

This report presents evidence that polymorphonuclear leucocytes (PMN) from chronic granulomatous disease (CGD) patients, who are defective in oxidative metabolism, are capable of inhibiting in vitro multiplication of Plasmodium falciparum . Using a microtitre in vitro inhibition assay, we incubated various numbers of peripheral blood neutrophils from CGD patients and from normal individuals with P. falciparum isolate F32 in the in vitro culture system. Inhibition of parasite growth by neutrophils was determined after 48 h of culture. At PMN to erythrocyte ratio of 1:50 there was an inhibition of parasite growth of 57% by normal neutrophils and 39% to 68% by CGD cells. When the neutrophils were stimulated by phorbol myristate acetate, both cell types enhanced inhibition of parasite growth. These findings indicate that the oxygen-independent systems of human neutrophils are involved in parasite destruction. Constituents of neutrophil granules such as acid hydrolases, lactoferrin, and cationic proteins could be regarded as potential mediators of parasite destruction.     

Generation of Reactive Oxygen Radicals by Human Phagocytic Cells Activated by Plasmodium falciparum

The role of monocytes, macrophages and neutrophils in killing malaria parasites is well documented, and their involvement in malaria pathology has been suggested. However, the underlying mechanisms are not clear. The present study reports on the role of P. falciparum-parasitized erythrocytes, free merozoites, and culture supernatant antigens in the generation of reactive oxygen radicals by human peripheral blood monocytes and neutrophils. Blood neutrophils and monocytes obtained from healthy individuals were isolated by density gradient separation. A human isolate of P. falciparum was grown in continuous culture. Parasitized erythrocytes and free merozoites were prepared from synchronized cultures. Soluble antigens from culture supernatants were purified by affinity chromatography using CNBr-Sepharose 4B columns bound to specific IgG. Oxidative burst response of neutrophils and monocytes were determined by oxygen consumption, superoxide production, and chemiluminescence. It was found that P. falciparum merozoites and the soluble antigens were capable of activating neutrophils and monocytes in vitro and resulting in the production of oxygen radicals by these cells. In conclusion, these findings demonstrate that malaria antigens are able to activate normal human blood phagocytes and result in generation of oxygen radicals by these cells. The released oxygen radicals can then contribute to both the destruction of the parasite and the pathology of malaria.     

Effector cells involved in nonspecific and antibody-dependent mechanisms directed against Plasmodium falciparum blood stages in vitro.

We have evaluated in in vitro conditions the possible cooperative effect of antimalarial antibodies with several human blood cell types. When used alone, immunoglobulin G from African adults who had reached a state of premunition against malaria was found to have no or very limited direct effect on invasion and multiplication of P. falciparum asexual blood stages. In contrast, these antibodies induced a marked specific inhibition of parasite growth in the presence of normal blood monocytes, and the inhibition did not appear to be strain dependent. No similar antibody-dependent cellular inhibitory effect was found using human blood polymorphonuclear leukocytes, lymphocytes, platelets, or adherent spleen cells. However, these cells could all exert in vitro some non-antibody-dependent inhibitory effect when present at high effector/target cell ratios.     

Identification and purification of glucose phosphate isomerase of Plasmodium falciparum.

The multiplication of malaria parasites within red blood cells is energy dependent. Since these parasites lack a functional tricarboxylic acid cycle, the energy needs of the parasite are met by anaerobic glycolysis of exogenous glucose. High levels of glycolytic enzymes such as fructose-1,6-diphosphate aldolase, phosphoglycerate kinase and pyruvate kinase have been detected in infected erythrocytes. Here we report a 4-9 times increase in glucose phosphate isomerase (GPI) activity of infected erythrocytes over that of normal erythrocytes. This increase is of parasitic origin, as additional enzyme bands were observed in lysates of infected erythrocytes. The expression of GPI parallels parasite maturation and reaches a maximum at the trophozoite/schizont stage. Two distinct but closely related activity patterns consisting of 3-4 GPI isoenzymes (not shown in normal erythrocytes) with neutral to weakly acidic isoelectric points were observed in 6 P. falciparum isolates tested by isoelectric focusing. The purified P. falciparum GPI has an apparent size of 66 kDa. No size variation was observed in the 6 P. falciparum isolates studied. Furthermore, antiserum raised against this protein in BALB/c mice specifically inhibits parasite encoded GPI activity while no effect was observed on host enzyme activity.     

Clinical correlates of in vitro Plasmodium falciparum cytoadherence.

To determine whether isolates of Plasmodium falciparum have intrinsically different cytoadherent properties and whether these differences contribute to the clinical severity of human falciparum malaria, we studied the cytoadherence to C32 melanoma cells in vitro of 59 parasite isolates from patients with naturally acquired infections in Thailand. Parasitized erythrocytes adhere to these melanoma cells principally via the glycoprotein CD36, which is also expressed on most vascular endothelium. In vitro cytoadherence was significantly greater for isolates from patients with biochemical evidence of severe malaria. The cytoadherent properties of P. falciparum parasites may thus be a virulence factor in human falciparum malaria. However, there was no correlation between the degree of in vitro cytoadherence and cerebral symptoms, which suggests that other receptors and/or host factors may be important in the adherence of malaria parasites to cerebral vascular endothelium. The cytokines tumor necrosis factor, interleukin-1, and gamma interferon, which have been implicated in the pathogenesis of cerebral malaria and are known to promote intercellular adhesion in other systems, did not enhance the cytoadherence of P. falciparum isolates to C32 melanoma cells.     

BDA-410: a novel synthetic calpain inhibitor active against blood stage malaria

Falcipains, the papain-family cysteine proteases of the Plasmodium falciparum, are potential drug targets for malaria parasite. Pharmacological inhibition of falcipains can block the hydrolysis of hemoglobin, parasite development, and egress, suggesting that falcipains play a key role at the blood stage of parasite life cycle. In the present study, we evaluated the anti-malarial effects of BDA-410, a novel cysteine protease inhibitor as a potential anti-malarial drug. Recombinant falcipain (MBP-FP-2B) and P. falciparum trophozoite extract containing native falcipains were used for enzyme inhibition studies in vitro. The effect of BDA-410 on the malaria parasite development in vitro as well as its anti-malarial activity in vivo was evaluated using the Plasmodium chabaudi infection rodent model. The 50% inhibitory concentrations of BDA-410 were determined to be 628 and 534nM for recombinant falcipain-2B and parasite extract, respectively. BDA-410 inhibited the malaria parasite growth in vitro with an IC(50) value of 173nM causing irreversible damage to the intracellular parasite. In vivo, the BDA-410 delayed the progression of malaria infection significantly using a mouse model of malaria pathogenesis. The characterization of BDA-410 as a potent inhibitor of P. falciparum cysteine proteases, and the demonstration of its efficacy in blocking parasite growth both in vitro and in vivo assays identifies BDA-410 is an important lead compound for the development of novel anti-malarial drugs.     

Assessment of immune phagocytosis of Plasmodium falciparum infected red blood cells by human monocytes and polymorphonuclear leukocytes. A method for visualizing infected red blood cells ingested by phagocytes

A method is described for the visualization of red blood cells infected with Plasmodium falciparum ingested by monocytes or polymorphonuclear leukocytes (PMN) after in vitro incubation. Smears were stained with peroxidase followed by 4,6-diamino-2-phenylindole (DAPI) staining specific for DNA. Monocytes or PMN were identified under normal illumination by the peroxidase stain and the nuclei of these cells as well as the parasites were identified by means of the DAPI stain with ultraviolet light. Using this method we found that monocytes and PMN from normal blood donors preferentially phagocytose plasmodium falciparum infected red blood cells in the presence of sera from subjects living in areas endemic for malaria.     

The A/T-specific DNA alkylating agent adozelesin inhibits Plasmodium falciparum growth in vitro and protects mice against Plasmodium chabaudi adami infection

There is an urgent need for new anti-malarial drugs to combat the resurgence of resistance to current therapies. To exploit the A/T richness of malaria DNA as a potential target for anti-malarial drugs we tested an A/T-specific DNA synthesis inhibitor, adozelesin, for activity against Plasmodium falciparum in vitro and Plasmodium chabaudi adami in mice. Adozelesin is a DNA alkylating agent that exhibits specificity for the motif A/T, A/T and A. In P. falciparum 3D7 cultures, adozelesin acts as a powerful inhibitor of parasite growth (IC(50) of 70 pM) and is equally potent at killing the drug-resistant strains FCR3 and 7G8. Using a real-time PCR assay, we show that treatment with adozelesin in vitro results in damage of P. falciparum genomic DNA. In synchronized cultures, adozelesin exhibits a concentration-dependent effect on parasitemia and on the development of parasites through the asexual cycle. In asynchronous cultures, parasites arrest at all stages of the asexual cycle suggesting that adozelesin exerts other anti-parasitic effects in addition to inhibiting DNA replication. These anti-parasite effects are irreversible since cultures exposed to adozelesin for more than 6h fail to recover upon removal of the drug. Furthermore, adozelesin is very effective at suppressing malaria infection in vivo; growth of P. c. adami DK in mice was highly impaired by a single injection of adozelesin (25 microg/kg) at 4 days post-infection. These results demonstrate that adozelesin irreversibly blocks parasite growth in vitro and suppresses parasite infection in vivo, suggesting that A/T-specific DNA damaging agents represent a new class of compounds with potential as anti-malarials.     

Specific antibody-dependent cellular cytotoxicity in human malaria.

A micromethod for the study of specific antibody-dependent cellular cytotoxicity (ADCC) in human malaria is described, using cultured, asexual Plasmodium falciparum parasites as viable target cells. Lymphocytes from children with acute malaria, uninfected immune adult Gambians and adult Gambians infected with P. falciparum were capable of killing P. falciparum in vitro in the presence of malaria antibody. A parasite growth-promoting factor, produced by lymphocytes in non-immune serum and at a lymphocyte--parasite ratio of 10:1, in immune serum, was found to produce three-fold increases in growth of P. falciparum. The mechanisms by which ADCC may occur are also discussed.     

Phagocytosis does not play a major role in naturally acquired transmission-blocking immunity to Plasmodium falciparum malaria

Phagocytosis of Plasmodium falciparum sexual stages in vitro and within the mosquito midgut was assayed in order to assess its role in transmission-blocking immunity to malaria. Both monocytes/macrophages (MM) and polymorphonuclear neutrophils (PMN) phagocytosed malarial gametes in vitro, but levels of phagocytosis were low. Intraerythrocytic gametocytes were not susceptible to phagocytosis. In vitro phagocytosis was positively correlated with levels of antibodies against the gamete surface proteins Pfs230 and Pfs48/45. Immunoglobulin G (IgG) subclass analysis revealed that phagocytosis was correlated with levels of antigamete IgG1. In vivo membrane-feeding experiments were performed in the presence of both pooled and individual malaria immune sera. The phagocytic process proceeded less efficiently in vivo than in vitro, which may be related to the lower ambient temperature (26 degrees C, compared with 37 degrees C). Finally, although we found a correlation between the ability of a serum to promote phagocytosis in vitro and the presence of antibodies against transmission-blocking target antigens, we were unable to demonstrate a role for MM- or PMN-mediated phagocytosis in reduction of infectivity of the malarial parasite to mosquitoes.     

Antigenic diversity of Plasmodium falciparum and antibody-mediated parasite neutralization

The malaria parasite Plasmodium falciparum, causing the most severe form of the disease in humans, is characterized by a broad antigenic diversity between different strains and isolates of the parasite. The antigenic diversity reflects on the one hand polymorphisms in allelic gene products and, on the other hand, antigenic variation as a result of expression of alternative genes in multigene families. Using selected polymorphic regions in two merozoite surface antigens, a method for genotyping P. falciparum parasites has been developed. This has resulted in new information on the clonal multiplicity and dynamics of parasite populations. Observations from in vivo and in vitro studies have identified many potential parasite-neutralizing immune responses and several of the target antigens are being explored as vaccine candidates. Studies of antibody-mediated neutralization of parasites in P. falciparum in vitro cultures, with or without leukocytes as effector cells, have been instrumental in identifying potential target antigens for protective immunity and for elucidation of the effects of immune pressure on the dynamics of parasite populations and their antigenic plasticity.     

Protection by alpha-thalassaemia against Plasmodium falciparum malaria: modified surface antigen expression rather than impaired growth or cytoadherence.

We have attempted to determine the cellular mechanism by which alpha-thalassaemia may protect against Plasmodium falciparum malaria. Invasion and development of P. falciparum in the microcytic red cells of two-gene deletion forms of alpha-thalassaemia when measured morphologically or by [3H]hypoxanthine incorporation were normal compared to controls. Normal invasion rates were also observed following schizogony in thalassaemic red cells. Neither the addition of the oxidant menadione, 30% oxygen, nor modified medium, produced differential damage to parasites within thalassaemic cells. Furthermore, there were no significant differences in the binding of P. falciparum-parasitized alpha-thalassaemic and normal cells to C32 melanoma cells in vitro. However, when neoantigen expression on the surface of infected thalassaemic cells was estimated using a quantitative radiometric antiglobulin assay, clear differences were observed. It was found that alpha-thalassaemic cells bound higher levels of antibody from serum obtained from individuals living in a malaria endemic area than control normal red cells. The binding ratio for thalassaemic compared with controls was 1.69 on a cell-for-cell basis, and 1.97 when related to surface area. The binding of antibody from immune serum increased exponentially during parasite maturation. We also found increased binding of naturally occurring antibody present in non-immune serum to parasitized thalassaemic red cells which also increased during parasite maturation. We conclude that the protection afforded by thalassaemia against malaria may not reside in the ability of parasites to enter, grow or cytoadhere to endothelium in such cells, but may be related to immune recognition and subsequent clearance of parasitized red cells.     

Protection against malaria in Aotus monkeys immunized with a recombinant blood-stage antigen fused to a universal T-cell epitope: correlation of serum gamma interferon levels with protection.

The major surface antigen p190 of the human malaria parasite Plasmodium falciparum contains nonpolymorphic, immunogenic stretches of amino acids which are attractive components for a subunit vaccine against malaria. One such polypeptide, termed 190L, is contained in the 80-kDa processing product of p190, which constitutes the major coat component of mature merozoites. We report here that immunization of Aotus monkeys with 190L gives only poor protection against P. falciparum challenge. However, addition by genetic engineering of a universal T-cell epitope (CS.T3) to 190L improved immunity, and as a result three of four monkeys were protected following challenge infection with blood-stage parasites. Neither antibody against the immunizing antigens or against blood-stage parasites nor the capacity of the monkeys' sera to inhibit in vitro parasite invasion correlated with protection. However, in contrast to sera from nonprotected monkeys, sera from protected animals contained elevated levels of gamma interferon. These results suggest that gamma interferon is directly or indirectly involved in the process of asexual parasite control in vivo.     

Gene disruptions demonstrate independent roles for the four falcipain cysteine proteases of Plasmodium falciparum

Erythrocytic stages of the malaria parasite Plasmodium falciparum express four related papain-family cysteine proteases, termed falcipains. Falcipain-2 and falcipain-3 are food vacuole hemoglobinases, but determination of the specific roles of these and other falcipains has been incomplete. To better characterize biological roles, we attempted disruption of each falcipain gene in the same strain (3D7) of P. falciparum. Disruption of falcipain-1, falcipain-2, and falcipain-2' was achieved. In each case knockouts multiplied at the same rate as wild-type parasites. The morphologies of erythrocytic falcipain-1 and falcipain-2' knockout parasites were indistinguishable from those of wild-type parasites. In contrast, consistent with previous results, falcipain-2 knockout trophozoites developed swollen, hemoglobin-filled food vacuoles, indicative of a block in hemoglobin hydrolysis and were, compared to wild-type parasites, twice as sensitive to cysteine protease inhibitors and over 1000 times more sensitive to an aspartic protease inhibitor. The falcipain-3 gene could not be disrupted, but replacement with a tagged functional copy was readily achieved, strongly suggesting that falcipain-3 is essential to erythrocytic parasites. Our data suggest key roles for falcipain-2 and falcipain-3 in the development of erythrocytic malaria parasites and a complex interplay between P. falciparum cysteine and aspartic proteases.     

Treatment and control of mycoplasma contamination in <Emphasis Type="Italic">Plasmodium falciparum</Emphasis> culture

A comparative efficacy of four antibiotics, plasmocin (macrolid), Biomyc-1, -2, (tetracycline), and Biomyc-3, and Mycoplasma Removing Agent (quinolone derivatives) was determined for elimination of mycoplasma from Plasmodium falciparum culture. Presence of mycoplasma was detected using enzyme-PCR-based mycoplasma detection kit and survival of malaria parasite was determined in Giemsa's stained smear made from treated and untreated cultures. It was observed that a combination of Biomyc-1 and -2 killed malaria parasites within 24 h, whereas plasmocin and Biomyc-3 caused slow death of malaria parasite stretched over a period of 6 days. The only compound which did not kill malaria parasite and eradicated mycoplasma from P. falciparum culture was observed to be MRA.     

Malaria antigen-specific T-cell responsiveness during infection with Plasmodium falciparum.

Protective immunity against Plasmodium falciparum develops only after several years of repeated exposure to the malarial parasite. We therefore investigated the possibility that acute malaria was associated with malarial antigen-specific immunosuppression. Peripheral lymphocytes of West Africans with and without P. falciparum infections were tested for their in vitro proliferative responses to a preparation of P. falciparum antigen. There was no significant difference between the magnitude of the proliferative response of lymphocytes from infected as compared to normal Africans, although the responses from both African groups were significantly higher than responses from a group of European controls. Furthermore, no soluble inhibitor of antigen-specific proliferation was present in plasma of infected patients. These observations strongly suggest that if the sluggish development of protective immunity in malaria is based upon infection-related immunosuppression, this occurs without affecting the proliferative responsiveness of specific sensitized, circulating T cells. Preliminary observations also indicate that Europeans residing in Africa and taking malaria prophylaxis may acquire sensitized T cells without experiencing clinically apparent infections.     

Multicenter study to evaluate the OptiMAL test for rapid diagnosis of malaria in U.S. hospitals

More than 1,000 cases of malaria are diagnosed each year in the United States. Reported numbers, however, may be artificially low because many clinicians fail to consider the diagnosis on presentation, U.S. hospital laboratory technologists have very limited experience in detecting and identifying malaria parasites, and reporting of malaria to state health departments is sporadic in many states. In this study, a rapid malaria diagnostic test, the OptiMAL test (DiaMed; under license from Flow Inc., Portland, Oreg.) was evaluated in six U.S. hospitals and compared with results of microscopy. The OptiMAL test is a 15-min rapid immunochromatographic test that both identifies and differentiates Plasmodium falciparum from non-P. falciparum malaria parasites on the basis of the detection of parasite lactate dehydrogenase in a drop of patient blood. A total of 216 specimens from patients suspected of having malaria were tested. Results indicated that 43 samples (20%) were positive for malaria parasites by microscopy (32 P. falciparum, 11 non-P. falciparum) while 42 (19%) were positive by OptiMAL (31 P. falciparum, 11 non-P. falciparum). The sensitivity of the OptiMAL test was 98%; its specificity was 100%, with positive and negative predictive values of 100 and 99%, respectively. Participating hospital physicians and laboratory directors independently reported that the OptiMAL rapid malaria test was accurate, easy to use, and well accepted by those working in their diagnostic laboratories. The overall conclusion was that integration of the OptiMAL rapid malaria test into the U.S. health care infrastructure would provide an important and easy-to-use tool for the timely diagnosis of malaria.     

Anti-malarial activity of leaf-extract of hydrangea macrophylla, a common Japanese plant

To find a new anti-malarial medicine derived from natural resources, we examined the leaves of 13 common Japanese plants in vitro. Among them, a leaf-extract of Hydrangea macrophylla, a common Japanese flower, inhibited the parasitic growth of Plasmodium falciparum. The IC50 of Hydrangea macrophylla leaf extract to Plasmodium falciparum was 0.18 microg/ml. The IC50 to NIH 3T3-3 cells, from a normal mouse cell line, was 7.2 microg/ml. Thus, selective toxicity was 40. For the in vivo test, we inoculated Plasmodium berghei, a rodent malaria parasite, to ddY mice and administered the leaf-extract of Hydrangea macrophylla (3.6 mg/0.2 ml) orally 3 times a day for 3 days. Malaria parasites did not appear in the blood of in the treated mice, but they did appear in the control group on day 3 or 4 after inoculation with the parasites. When leaf extract was administered to 5 mice 2 times a day for 3 days, malaria parasites did not appear in 4 of the mice but did appear in 1 mouse. In addition, the leaf-extract was administered orally 3 times a day for 3 days to Plasmodium berghei infected mice with a parasitemia of 2.7%. In the latter group, malaria parasites disappeared on day 3 after initiating the treatment, but they appeared again after day 5 or 6. Although we could not cure the mice entirely, we confirmed that the Hydrangea macrophylla leaf extract did contain an anti-malarial substance that can be administered orally.     

Molecular analysis of Plasmodium falciparum infections in man]

Plasmodium falciparum diversity has been analysed in two Senegalese villages with different transmission conditions and distinct kinetics of immunity acquisition. A very large allelic polymorphism was observed in both villages, with a similar number of alleles but quite distinct allelic frequencies, indicating a substantial micro-geographical heterogeneity of malaria parasite populations. In addition, the molecular characteristics of the infections differed in both villages. As in most endemic areas, many infected subjects carry multiple parasite clones. In Dielmo, the number of distinct clones hosted decreases at the age of acquisition of an efficient immunity. There was no influence of age on the number of clones hosted in Ndiop where adults experience clinical attacks. This indicates that complexity reflects acquired immunity. The precise longitudinal follow-up of parasitaemia, clinical signs and parasite genetic characteristics showed a rapid turn over of parasite populations in the peripheral blood during the transmission season, suggesting that immunity does not prevent infection but restricts multiplication of numerous genotypes at the erythrocytic stage. Clinical malaria occurs after a rapid, apparently unrestricted growth of recently inoculated parasites. The successive clinical attacks experienced by children are associated with genotypes different for each attack and different from those that the child carried during preceding asymptomatic phases. These data indicate that parasite diversity contributes to the pathology of infection and that control of parasite density, which is at least in part strain-specific, is an essential element of protection against malaria clinical attacks.     

Killing of Plasmodium falciparum by cytokine activated effector cells (neutrophils and macrophages)

Macrophages display natural antibody independent killing of asexual blood stages of Plasmodium falciparum in vitro. In contrast, the neutrophil killing of P. falciparum requires the presence of antibodies. Cytokines such as TNF alpha have very little effect on the macrophage-induced antiplasmodial activity, but significantly increase the damage of parasites by neutrophils. Cytokines, TNF alpha, IFN-gamma and TNF beta at very high concentrations were not toxic to P. falciparum in culture. It is postulated that the basis for cytokine modulated antiplasmodial activity of leukocytes is increased expression of Fc and complement receptors, which leads to a more efficient interaction between the parasite and neutrophils. It is also postulated that the parasite evades natural macrophage killing mechanisms by inducing factors which suppress this macrophage activity. Cytokine inhibitors may be induced during the course of a malarial infection. These could be involved in attempts to attain a balance between the host and the parasite, by protecting the parasite from the damaging effect of the immune system and protecting the host from the deleterious effects of cytokines.